Supplementary Table 1. Immunological Characteristics of PatientsPatients P1.1 P1.3 P1.4 P2.1 P2.2 P3.3 mother

P2.1mother

P2.2Age (years) 8 10 40 23 11 36 47 35Leukocyte

countsa7500

(4400-9500)5900

(4400-9500)n.d 5100

(4400-9500)4800

(4400-9500)4900

(4400-9500)4900

(4400-9500)11500

(4400-9500)Polynuclear

countsa3900

(1900-8000)3700

(1900-8000)n.d 2500

(1900-8000)2000

(1900-8000)2700

(1900-8000)3500

(1900-8000)8200

(1900-8000)Monocyte

countsa225

(200-600)400

(200-600)n.d 500

(200-600)400

(200-600)600

(200-600)200

(200-600)500

(200-600)Lymphocyte

countsa3300

(1900-3700)1800

(1900-3700)n.d 2100

(1000-2800)2400

(1900-3700)1600

(1000-2800)1200

(1000-2800)2800

(1000-2800)T

lymphocytesa

(CD3)

2740(1200-2600)

1556(1200-2600)

n.d 1174(700-2100)

1962(1200-2600)

1066(700-2100)

633(700-2100)

2322(700-2100)

CD4lymphocytesa

1122(650-1500)

306(650-1500)

n.d 661(460-1230)

646(650-1500)

677(460-1230)

464(460-1230)

1465(460-1230)

CD45RO/CD4(%)

n.d n.d n.d 26(16-34)

12.2(13-30)

n.d 42.2(16-34)

26.6(16-34)

CD8lymphocytesa

1650(370-1100)

1044(370-1100)

n.d 687(190-850)

1396(370-1100)

599(190-850)

325(190-850)

890(190-850)

CD45RO/CD8(%)

n.d n.d n.d 17.6(2.6-22.9)

26.5(4-21)

n.d 11.2(2.6-22.9)

7(2.6-22.9)

Blymphocytesa

(CD19)

264(270-860)

216(270-860)

n.d 517(110-570)

125(270-860)

67(110-570)

196(110-570)

302(110-570)

CD27/CD19(%)

n.d 40.3(10-50)

n.d 15.6(10-50)

13.7(10-50)

62.1(10-50)

57.9(10-50)

42.2(10-50)

NK cellsa

(CD16/CD56)297

(100-480)28

(100-480)n.d 436

(92-420)51

(100-480)215

(92-420)235

(92-420)87

(92-420)PHA-T cell

Proliferationb88

(>40)119

(>40)n.d n.d n.d n.d n.d n.d

IgGc 12.8(5.7-13.2)

4.17(5.7-13.2)

2.5(9.2-14.8)

11.7(9.2-14.8)

9.71(5.7-13.2)

6.04(9.2-14.8)

n.d n.d

IgAc 0.58(0.65-2.4)

0.08(0.65-2.4)

0.49(1.42-2.62)

2.99(1.42-2.62)

3.42(0.65-2.4)

1.29(1.42-2.62)

n.d n.d

IgMc 0.75(0.6-1.75)

2.05(0.6-1.75)

1.38(0.88-1.84)

0.43(0.88-1.84

1.2(0.6-1.75)

0.27(0.88-1.84)

n.d n.d

Parameters have been checked at least two times and found to be stable over the time.n.d : not determinedAge matched control values are indicated in italics in bracketsaNumbers of cells/mm3

bPhytohemagglutinin (PHA)-induced proliferations are expressed as couts per minute x10-3

cin mg.ml-1

Supplementary Figure 1. Expression of SAP in cells from affected individuals of XLP families without SAP mutations. Cell lysates from PHA blasts of healthy control individuals (Ctr.), patients (P1.3, P2.1 and P3.1) and a SAP-deficient patient (SAP) were analyzed by Western blotting with anti-SAP antibodies (upper panel). Immunoblot with anti-Fyn antibodies (lower panel) is shown as control of loading.

Supplementary Figure 2. Expression of genes surrounding XIAP, THOC2, STAG2 and SAP is normal in XIAP-deficient patients. The relative expression of THOC2, XIAP, STAG2, SAP and G3PDH was examined by RT-PCR in lymphocytes from two XIAP-deficient patients (P1.1 and P2.1) and two healthy control individuals (Ctr.). Human G3PDH RT-PCRs are shown as normalization controls of the samples. Three fold-serial dilutions of cDNAs (indicated as 1, 0.3 and 0.1) were used for amplification of each gene. Products were resolved by 1% agarose gel electrophoresis and were detected by ethidium bromide staining.

cba

Supplementary Figure 3. Expression of XIAP in the different hemopoetic lineages (a) and lack of expression of truncated or altered XIAP proteins in cells from XIAP-deficient patients (b,c). a, Cells were purified as stated in the “Methods” section. Cell lysates from PBMCs, B cells, CD4 T cells (CD4+), CD8 T cells (CD8+), monocytes (monocytes), dendritic cells (DC), MAB11 NK T cells (NKT) and NK cells (NK). b and c, Cell lysates of PHA blasts from healthy control (Ctr.) and XIAP-mutated (P3.1, P2.1 and P1.3) individuals were electrophoresed in 15% (a) or in 8% (b) SDS-polyacrylamide gels and immunoblotted with anti-XIAP antibodies raised against the N-terminus of XIAP (H202). Immunoblot with anti-Ku80 antibodies is shown as control of loading.

Supplementary Figure 4. Analysis of NK cells from XIAP-deficient patients. a, The 2B4-mediated cytolytic activity is impaired in SAP-deficient NK cells (SAP) but not in XIAP-deficient NK cells. Lysis of L1210 target cells by NK cells from healthy control (C1 and C2), SAP-deficient (SAP1 and SAP2) or XIAP-deficient (P1.7 and P2.2) individuals that have been stimulated by anti-2B4, anti-CD16 or control antibodies (cont. Ab). b, Lysis of K562 target cells by NK cells from normal (C1, C2 and C3), SAP-deficient (SAP1 and SAP2) or XIAP-deficient (P1.1 and P2.2) individuals. c, Percentages of NK cells (CD56+/CD3-) in PBMCs of blood samples from Ctr., SAP, XIAP and SIM individuals. Bars correspond to mean values of each group of NK cell percentages. Values of means s.d.: Ctr, 6.24.8 ; SAP, 2.62.5 ; XIAP, 3.472.9 ; SIM, 3.23.8. SIM =Sporadic Severe Infectious Mononucleosis.

b

C1P2.2C2 C3

P1.1SAP1

SAP2

% s

peci

fic ly

sis

0

20

40

60

80

a

0

20

40

60%

spe

cific

lysi

s

C2 P1.7 SAP1 SAP2P2.2 C1

cont. Abanti-2B4anti-CD16

0

5

10

15

Ctr. XIAP SIM(n=11) (n=8) (n=7)(n=8)

% o

f NK

cel

ls (P

BM

C)

SAP

c

b

Supplementary Figure 5. Analysis of NKT cells of female XIAP-mutated carriers. a, Comparison of the numbers of NKT cells (V24 TCR+ V11 TCR+) in CD3+ lymphocytes from two female XIAP-mutated carriers (Mothers) and their XIAP-deficient sons (Patients). NKT cells were analyzed after gating on CD3+ cells by the staining with anti-V24 and anti-V11 TCR antibodies. The XIAP patients tested are P2.1, P2.2. Relatives are indicated by a trait. Values of means s.d.: Mothers, 24317.7 ; Patients, 8121. b, c. Non-random X chromosome inactivation in NKT cells from a female XIAP-mutated carrier. X chromosome inactivation was examined by analyzing the methylation pattern of the human androgen-receptor gene (AR) b, X chromosome inactivation analysis in total blood cells from two female XIAP-mutated carriers and one control healthy female (Ctr.). Extracted DNA of blood from control (Ctr.) and XIAP-mutated carriers (mothers of P2.1 and P2.2) were digested (+) or not (-) with HpaII, a methylation-sensitive enzyme. Maternal and paternal polymorphic alleles of AR were then amplified by PCR from digested or non-digested DNAs. The PCR products were resolved by 5% polyacrylamide gel electrophoresis. The two major products amplified from the non-digested DNAs of the control and the XIAP-mutated carrier individuals correspond to the maternal and paternal polymorphic AR alleles. They are respectively indicated by an asterisk and an arrow. In HpaII-digested DNAs from the two XIAP-mutated carriers, the two AR alleles were unequally amplified by contrast to the non-digested DNAs and to the digested DNA from the control carrier indicating non-random X chromosome inactivation in the two XIAP-mutated carriers. c, X chromosome inactivation in the different blood cell populations including NKT cells. T lymphocytes (T), B lymphocytes (B), NK cells (NK), monocytes (Mono) and NKT lymphocytes (NKT) from one female XIAP-mutated carrier (mother of P2.2) were purified by FACS sorting. The analysis of the X chromosome inactivation was then analyzed in purified populations as stated in b. In HpaII-digested DNAs from the different blood cell populations, the two AR alleles were unequally amplified by contrast to the non-digested DNAs indicating non-random X chromosome inactivation.

a

Mothers Patients0

100

200

300

400

500

6

c

c

Anti-CD95 (ng ml-1) :

a

0

20

40

60

80

100

% o

f ind

uced

apo

ptos

is

P2.1C1

5 10 15 20 5 10 15 20

P1.3C3

P3.3C2

5 10 15 20

0

P3.3C.2

P2.1C.1

P1.1C.3

10

20

30

% o

f ind

uced

apo

ptos

is

b

P3.3C.2

P1.3

C.3

P2.1C.1

0

20

40

60

80

100

% o

f ind

uced

apo

ptos

is

Fas+

Fas-L

P2.1C.1

Supplementary Figure 6. Enhanced apoptosis of XIAP-deficient T lymphocytes to apoptotic stimuli. a, Induced apoptosis of T cell blasts from control healthy (C1, C2 and C3) and XIAP-deficient individuals (P3.1, P2.1 and P1.3) that were stimulated with various concentrations of anti-CD95 antibodies as indicated. b, Activation-induced cell death (AICD) of T cells from normal (C) and XIAP-deficient individuals (P). T cell blasts were stimulated with anti-CD3 antibodies and analyzed for apoptosis. c, Induced apoptosis of T cells stimulated with a trimeric TRAIL molecule. Data are means of duplicates from one representative experiment.

c

Ctr. 1Ctr. 2Ctr. 3P1.1

Anti-CD95 (ng ml-1) :

% o

f ind

uced

apo

ptos

is

a

Time (days)

P1.1

% o

f GFP

-pos

itive

cel

ls

Time (days)

b Ctr.

Supplementary Figure 7. Expression of wild-type XIAP in XIAP-deficient EBV-transformed B cells allows their selective survival and expansion upon anti-CD95 stimulation. a, Enhanced apoptosis of XIAP-deficient EBV-transformed B cells to anti-CD95 apoptotic stimuli. EBV-transformed B cell lines derived from three healthy control individuals (Ctr.) and a XIAP-deficient patient, P1.1 (XIAP) were stimulated with various concentrations of anti-CD95 antibodies as indicated, and analyzed for apoptosis. b, EBV-transformed B cell lines from healthy control (Ctr.) and XIAP-deficient (XIAP) individuals were infected with the pTRIP-GFP-XIAP vector or the empty vector pTRIP-GFP. Infected cells with pTRIP-GFP-XIAP were then repeatedly stimulated with anti-CD95 (100ng/ml) during the culture as indicated by the arrows. The percentages of GFP-positive cells were determined by flow cytometry during the culture. c, Reconstituted expression of XIAP in XIAP-deficient EBV-transformed B cells. Cell lysates from control (Ctr.) and XIAP-deficient (XIAP) EBV-transformed B cells at day 25 of culture, infected with pTRIP-GFP, pTRIP-GFP-XIAP and treated or not with anti-CD95, or non-infected (/) were immunoblotted with anti-XIAP antibodies or anti-Ku80 antibodies as control of loading.